The mechanisms controlling the initiation of DNA replication in eukaryotes are of great interest since inappropriate DNA replication can cause uncontrolled cell growth (cancer), cell death, and abnormal development. Initiation of DNA replication at eukaryotic chromosomes requires that first the site of initiation be selected and then the two strands of DNA be separated to provide a single stranded DNA template for DNA polymerase action. Recently a complex of polypeptides was purified from yeast extracts that specifically recognizes yeast replication origins in vitro. This complex, known as the Origin Recognition Complex (ORC), was shown in subsequent genetic experiments to be essential for S. cerevisiae DNA replication in vivo. Combined, these in vitro and genetic data suggest that ORC acts to select the site of initiation of DNA replications at S. cerevisiae chromosomes. ORC does not, however, independently melt the origin DNA suggesting that additional co-factors are required for origin melting. I propose to use an assay for DNA melting to identify S. cerevisiae protein fractions that catalyze melting of origin DNA in the presence of purified ORC protein. Identified melting co-factors will be purified and further characterized to understand their role in origin melting. The origin sequence dependence and the role of ORC in origin melting will also be investigated.